Allorestricted peptide-specific T cells

ABSTRACT

The present invention is directed to a T cell receptor (TCR) recognizing antigenic peptides derived from tumor-associated antigen FMNL1/KW13 and being capable of inducing peptide specific killing of a target cell. The present invention is further directed to one antigenic peptides derived from tumor-associated antigen FMNL1/KW13, to an antigen specific T cell, comprising said TCR, to a nucleic acid coding for said TCR and to the use of the antigen specific T cells for the manufacture of a medicament for the treatment of malignancies characterized by overexpression of FMNL1/KW13.

CROSS-REFERENCE TO RELATED APPLICATION

The instant application is a National Stage Application filed under 35U.S.C. §371 of PCT International Patent Application No.PCT/EP2007/056029, filed Jun. 18, 2007. The disclosure of PCTInternational Patent Application No. PCT/EP2007/056029 is herebyincorporated by reference in its entirety.

The present invention is directed to T cell receptors (TCR) recognizingantigenic peptides derived from tumor-associated antigen FMNL1/KW13 orthe murine homolog FRL and being capable of inducing peptide specifickilling of a target cell. The present invention is further directed toantigenic peptides derived from tumor-associated antigen FMNL1/KW13, toan antigen specific T cell, comprising said TCR, to a nucleic acidcoding for said TCR and to the use of the antigen specific T cells forthe manufacture of a medicament for the treatment of malignanciescharacterized by overexpression of FMNL1/KW13. The present invention isfurther disclosing a method of generating antigen specificallorestrictive T cells.

BACKGROUND OF THE INVENTION

The present invention concerns the field of immunotherapies in B cellneoplasms, in particular B cell lymphoma, including acute and chroniclymphocytic leukemias.

The incidence of B cell neoplasms is increasing, especially in WesternCountries (NCI, SEER). Most cases of low grade lymphoma are diagnosed atadvanced stages where no curative standard therapy is available. Inchronic lymphocytic leukemia (CLL), there is still no establishedcurative therapy available at any time point of diagnosis. In contrast,patients with high grade lymphoma or acute lymphoblastic leukemiabenefit from intensive and potentially curative chemotherapy regimens.However, longterm prognosis is unfavourable for adult patients withrecurrent disease. Thus, novel therapies are needed.

Immunotherapy has been shown to be highly successful in low gradelymphoma including CLL. There are target specific therapies usingmonoclonal antibodies which are directed against surface antigens asCD20 (Rituximab) and CD52 (Alemtuzumab) (1,2). However, tumor escapeoccurs due to down regulation of the target antigen from the cellsurface. T cells are able to recognize peptides associated to MHCmolecules derived from extra- and intracellular antigens. T cells arecurrently applied associated to allogeneic stem cell transplantation andresponsible for a Graft versus leukemia effect (GvL effect) which hasbeen shown to be present in different B cell neoplasms (3-5). However,one major disadvantage of this approach is Graft versus host disease(GvHD) due to transferred T cells unspecifically recognizing allogeneicMHC molecules. Using allorestricted peptide-specific T cells it might bepossible to circumvent central tolerance against tumor associated selfpeptides and simultaneously dissociate beneficial graft versus leukemiaeffect from detrimental graft versus host disease (6,7).

Krackhardt et al., “Identification of tumor associated antigens inchronic lymphocytic leukaemia by SEREX”, Blood September 2002, vol. 100,no. 6, pages 2123-2131, describe the identification of KW13/FMNL1. Here,peptide-specific autologous T cells were generated recognizing onesingle FMNL1-derived peptide (TLLHYLVKV; SEQ ID NO: 21). However, itturned out that these T cells did not recognize tumor cells as describedin the paper. Thus, it is likely that the specific peptide is not anepitope presented on the surface of CLL cells. Moreover, the specificpeptide has not been listed. In addition, no specific TCR has beendescribed.

The identification of an antigenic epitope, however is of fundamentalimportance to use a specific antigen as target for a therapeuticapproach as tumor cells can be only targeted if the specific peptide ispresented on the surface of a cell. Given a protein length of 1100 aminoacids, there are 1091 different nona-peptides which can be used forpulsing and most of them may not bind to the restriction element and/orare not produced by the immune proteasome, thus they are mostly notpresented on the surface of a tumor cell.

Mayr et al., “Transduction of CLL cells by CD40 ligand enhances anantigen-specific immune recognition by autologous T cells”, BloodNovember 2005, vol. 106, no. 9, compare the functional reactivity of Tcell lines generated by stimulation with CD40L-AAV-transduced CLL cells,GFP-AAV-transduced CLL cells as well as native CLL cells and CLL cellsstimulated by CD40-Ligand. Their read-out is an ELISPOT-assay where theyuse peptide-pulsed APC using different peptides for pulsing. They claimthat they have generated antigen-specific T cells. However, severalimportant controls are missing in order to prove the significance of thepeptides used for detection of specific antigen recognition, as forexample peptides and well characterized T cells for positive andnegative controls, peptide dilution and tetramer-staining of T celllines. As CD40L-activated CLL cells may prime for many antigens andpeptide-pulsed CLL cells can express many other antigens,peptide-specificity is not shown in the manuscript. Moreover, nospecific peptide is mentioned concerning KW13. The authors merelymention “KW13” in the methods section. KW13 is actually not a peptidebut the whole FMNL1 protein. In addition, no specific TCR has beendiscovered and described. Thus regarding Mayr et al., no precise peptidehas been described concerning KW13/FMNL1, no peptide-specificity hasbeen proven, no epitope has been discovered, no specific TCR was shown.This is exactly the critical development which has been undertaken inour last work and which motivates us for the patent claims.

SUMMARY OF THE INVENTION

It one object of the present invention to generate allorestrictive Tcells that bear TCR that have the capacity to recognize theirMHC-peptide ligands on tumor cells. Furthermore, it is an object of theinvention to provide a T cell based pharmaceutical composition that canbe used for treating a patient suffering from lymphoma without a risk ofgraft-versus-host-disease (GVHD).

These objects are achieved by the subject-matter of the independentclaims. Preferred embodiments are set forth in the dependent claims.

The inventors have generated allorestricted peptide-specific T cellswith specificity against a defined peptide derived from the tumorassociated antigen FMNL1/KW13. For example, it could be demonstrated (I)the identification of specific T cell receptor chains which areallo-HLA-A2 restricted and specific for our selected peptide RLPERMTTL(SEQ ID NO: 2) as well as the murine homolog RLPERMNTL (SEQ ID NO: 5)derived from the murine FMNL1 homologous protein and (II) that theselected peptide RLPERMTTL (SEQ ID NO: 2) derived from FMNL1/KW13 is anatural presented epitope overexpressed on malignant tissue shown byspecific cytotoxicity against lymphoma cell lines, other malignant celllines and primary tumor material of patients with chronic lymphocyticleukaemia and acute lymphoblastic leukemia. These T cells will behelpful for the development of immunotherapies against malignantlymphoma.

In healthy tissue, FMNL1/KW13 is almost exceptionally expressed inleukocytes. FMNL1/KW13-specific allorestricted T cells, therefore, arealso useful in the development of immunotherapeutic strategies againstrheumatoid diseases.

Using the present approach, allorestricted peptide-specific T cells withspecificity against the selected peptides which also show specificcytotoxicity against malignant cell lines derived from lymphoma, renalcell carcinoma, melanoma, breast cancer, as well as cells from patientswith chronic lymphocytic leukaemia, acute lymphoblastic leukaemia cellsand also activated lymphocytes could be generated. In contrast, healthytissue cell lines as fetal fibroblasts and fetal kidney cells were notrecognized (FIGS. 4-12).

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the invention provides a T cell receptor(TCR) recognizing antigenic peptides derived from tumor-associatedantigen FMNL1/KW13 or murine FRL and capable of inducing peptidespecific killing of a target cell.

More precise information regarding tumor-associated antigen FMNL1/KW13may be found in (8), including accession no., suggested function etc.

The term “TCR” as used in the present invention has the common meaning,which usually is attributed to that term in the pertinent field oftechnology. Thus, a rearranged T cell receptor (TCR) comprises a complexof two chains (α-chain and β-chain) containing a CDR3-region ofrearranged TCR VDJ genes mainly involved in the recognition of antigenicdeterminants (epitopes) represented in the MHC context. More detailedinformation can be found in “Immunobiology, the immune system in healthand disease”, Charles A. Janeway, et al, 5 ed. 2001 and other standardliterature.

An “antigenic peptide” as used herein is defined as comprising at leastone antigenic determinant, i.e. an epitope. The latter is a part of amacromolecule that is being recognized by the immune system, in thepresent case specifically by cytotoxic T cells.

Accordingly, the TCR of the present invention specifically recognizesone of the epitopes of any of SEQ ID NO: 1-5 and/or peptides/proteinscontaining same. It is noted that the epitope according to SEQ ID NO: 2turned out to be most preferred in the present invention since it is anatural presented epitope overexpressed on malignant tissue and the TCRrecognizing same effectively induced a specific cytotoxicity againstlymphoma cell lines. The present invention also provides an homologouspeptide to epitope of SEQ ID NO: 2, i.e. the murine homologue derivedfrom FRL.

It is noted that the invention is not restricted to the precise aminoacid sequences as defined herein, but also include variants of thesequences, for example deletions, insertions and/or substitutions in thesequence, which cause for so-called “silent” changes.

Preferably, such amino acid substitutions are the result ofsubstitutions which substitute one amino acid with a similar amino acidwith similar structural and/or chemical properties, i.e. conservativeamino acid substitutions.

Amino acid substitutions can be performed on the basis of similarity inpolarity, charges, solubility, hydrophobic, hydrophilic, and/oramphipathic (amphiphil) nature of the involved residues. Examples forhydrophobic amino acids are alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. Polar, neutral aminoacids include glycine, serine, threonine, cysteine, thyrosine,asparagine and glutamine. Positively (basic) charged amino acids includearginine, lysine and histidine. And negatively charged amino acidsinclude aspartic acid and glutamic acid.

The allowed degree of variation can be experimentally determined viamethodically applied insertions, deletions or substitutions of aminoacids in a peptide and testing the resulting variants for theirbiological activity as an epitope. In case of variation of the TCR-CDR3region specificity and function of the modified TCR can beexperimentally investigated by TCR expression in transduced cells or bypurified TCRs analyzed with surface plasmon resonance (e.g. Biacore).

An example of such a variant is the sequence of the murine homologue ofSEQ ID NO: 2. Whereas the human sequence is RLPERMTTL (SEQ ID NO: 2),the murine homologue is RLPERMNTL (SEQ ID NO: 5). This variant showedalso a very good recognition by TCR.

According to a further preferred embodiment, the TCR contains one ormore of the amino acids of SEQ ID NO: 9-14.

In a second aspect, the present invention provides an antigenic peptidederived from tumor-associated antigen FMNL1/KW13 being selected from oneof SEQ ID NO: 1-5.

However, also the epitopes of SEQ ID NO: 6-8 are contemplated herein.

According to a third aspect, the present invention provides an antigenspecific T cell, comprising a TCR as defined above.

Said T cell preferably is a T cell with effector cell characteristics,more preferably a cytokine producing T cell, a cytotoxic T cell orregulatory T cell, preferably CD4+ or CD8+ T cells. Most preferably, theT cell is an autologous T cell.

In a fourth aspect, the invention provides a nucleic acid coding for apart of a TCR (CDR3-region) as defined above. Respective sequences areprovided as SEQ ID NO: 15-20.

An additional aspect is directed to a vector, which comprises thenucleic acid coding for said TCR. This vector is preferably anexpression vector which contains a nucleic acid according to theinvention and one or more regulatory nucleic acid sequences. Preferably,this vector is a plasmid or a retroviral vector.

The invention further comprises a PBMC, which has been transformed withthe vector as defined above. This can be done according to establishedmethods such as those described in Engels et al., 2005 (9).

In a still further aspect, the present invention provides apharmaceutical composition, which comprises the T cells or PBMCs asexplained above and a pharmaceutically acceptable carrier.

Those active components of the present invention are preferably used insuch a pharmaceutical composition in doses mixed with an acceptablecarrier or carrier material, that the disease can be treated or at leastalleviated. Such a composition can (in addition to the active componentand the carrier) include filling material, salts, buffer, stabilizers,solubilizers and other materials, which are known state of the art.

The term “pharmaceutically acceptable” defines a non-toxic material,which does not interfere with effectiveness of the biological activityof the active component. The choice of the carrier is dependent on theapplication.

The pharmaceutical composition can contain additional components whichenhance the activity of the active component or which supplement thetreatment. Such additional components and/or factors can be part of thepharmaceutical composition to achieve synergistic effects or to minimizeadverse or unwanted effects.

Techniques for the formulation or preparation and application/medicationof active components of the present invention are published in“Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa.,latest edition. An appropriate application is a parenteral application,for example intramuscular, subcutaneous, intramedular injections as wellas intrathecal, direct intraventricular, intravenous, intranodal,intraperitoneal or intratumoral injections. The intravenous injection isthe preferred treatment of a patient.

According to a preferred embodiment, the pharmaceutical composition isan infusion or an injection or a vaccine.

According to a further aspect, the present invention is directed to theuse of the antigen specific T cells or PBMCs as explained above for themanufacture of a medicament for the treatment of malignanciescharacterized by overexpression of FMNL1/KW13, preferably chroniclymphocytic leukemia (CLL), or rheumatoid diseases.

In a further aspect, the invention provides a method of generatingantigen specific allorestrictive T cells comprising the steps of

a) providing a FMNL1/KW13 (or FRL) derived antigenic peptide as definedabove;

b) pulsing antigen presenting cells (APCs) with said peptide;

c) priming peripheral blood lymphocytes (PBLs) with said APCs;

d) selecting those T cells which are specific for the resultingMHC-peptide ligand.

The APC's are preferably selected from dendritic cells, activated Bcells, monocytes, macrophages, activated T cells, hematologicalmalignancies with antigen presenting capacities and/or EBV-transformedlymphoblastoid cell lines.

Dendritic cells (DC) are in particular preferred. Mature dendritic cells(DCs) express both MHC class I and class II molecules at high levels,along with a wide variety of costimulatory molecules, which provide themwith the full capacity to prime naïve T cells that have not encounteredantigen previously. They also have all the necessary genes/proteins thatallow them to process and present antigens form intracellular proteinsin their MHC class I and class II molecules. Thus, they are optimalantigen presenting cells (APCs) to use as stimulating cells forinduction of both CD4 and CD8 T cells responses.

The selection step d) is preferably performed by means of measuring thecytokine release of the T cells or other measures of T cell activation.For example, the activated T cells can be cloned as individual cells andfollowing expansion, the T cell clones can be analyzed for theirMHC-peptide specificity and those with the desired specificity can beselected for further use (10). Alternatively, soluble MHC-peptideligands in various forms, such as tetramers, can be marked with afluorescent label and incubated with the activated T cells. Those Tcells bearing TCR that interact with the tetramers can then be detectedby flow cytometry and sorted on the basis of their fluorescence (11).Furthermore, T cells can be stimulated for short periods of time withtumor cells to which they should react and their interferon gammasecretion detected by capture reagents, for example as published (12).

According to a preferred embodiment, the method of the invention furthercomprises the step of expanding the T cells selected in d) ex vivo. Thiscan be done by co-culturing the selected T cells with APC generated inthe same manner as used for their initial priming, adding new APC to theT cell cultures every 7-10 days and providing the cells with freshculture medium on a regular basis that contains supplementary cytokines,dependent upon the type of T cell that one is expanding.

The present invention in the following is illustrated by the Figures andExamples presented below, which in no way should be construed to belimiting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Fold expression of FMNL1/KW13 mRNA in normal tissue compared toskeletal muscle investigated by quantitative RT-PCR.

FIG. 2: Fold expression of FMNL1/KW13 mRNA in PBMC, native CLL samplesand acute leukaemia samples compared to skeletal muscle investigated byquantitative RT-PCR.

FIG. 3: Fold expression of FMNL1/KW13 mRNA in malignant cell linescompared to skeletal muscle investigated by quantitative RT-PCR.

FIG. 4: Enrichment of allorestricted KW13-PP2-specific T cells byTetramer sorting. Bulk cultures before sorting, enriched T cell linesand 4 cloned T cells are stained with anti-human CD8 APC andKW13-PP2-Tetramer-PE.

FIG. 5: Mini-Chromium-Assay to detect KW13-PP2-specific killing ofcloned T cells. ⁵¹Cr-labeled targets are T2 cells pulsed with thespecific peptide PP2 and unpulsed T2 cells as controls.

FIG. 6: Functional analysis of KW13-PP2 specific T cell lines andclones. Investigation of T cell affinity of 5 different T cell lines andclones in response to T2 cells pulsed with titrated concentrations ofthe specific peptide KW13-PP2.

FIG. 7: Functional analysis of KW13-PP2 specific T cell lines andclones. Bioplex-Multianalysis ELISA of PP2-SC22 in response to T2 cellspulsed with the specific peptide KW13-PP2, T2 cells pulsed with theFlu-peptide (MP58) and BJ18 lymphoma cells.

FIG. 8: Titration of the Effector:Target-Ratio with (a) PP2-SC13 aseffector cells and KW13-PP2-pulsed T2 cells as targets and (b) PP2-SC14as effector cells and BJ18 lymphoma cells as targets.

FIG. 9: Functional analysis of KW13-PP2 specific T cell lines andclones.

(a) Specific cytotoxicity of PP2-SC14 in response to pulsed T2 cells anddifferent malignant cell lines and primary healthy cell material and (b)specific cytotoxicity of PP2-PC1, PP2-SC14 and PP2-SC19 againstKW13-PP2-pulsed T2 cells, unpulsed T2 cells and acute lymphoblasticleukemia (ALL).

FIG. 10: Overview of specific cytotoxicity of cloned KW13-PP2-specific Tcells against malignant cell lines and primary tumor material.

FIG. 11: Overview of specific cytotoxicity of cloned KW13-PP2-specific Tcells against healthy PBMC subpopulations and cell line derived fromhealthy tissue.

FIG. 12: Specific cytotoxicity of PP2-SC22 against T2 cells pulsed withthe peptide FMNL1-PP2, the murine homologue FRL-PP1, a highly homologouspeptide derived from HDAC6 as well as native and CD40-activated CLLcells.

FIG. 13. Generation of the FMNL1-specific antibody. (A) Human FMNL1sequence (Swiss-Prot database, Accession No. O95466; SEQ ID NO: 25).Matched peptides detected by mass spectrometry analysis are shown (boldand framed) covering 24% of the protein. (B) Four different monoclonalantibody supernatants (5C9, 5B12, 6F2, 5A1) were tested in a Westernblot using 50 μg of cell lysates from T293 cells transfected withpCMV-FMNL1 (F) and pCMV-Vector alone (M).

FIG. 14. Peptide competition assay. Peptide candidates derived fromFMNL1 were investigated for their potential binding ability to HLA-A2.Therefore, different FMNL1-derived peptides were loaded on T2 cells andafterwards pulsed with the tyrosinase-derived peptide (Tyr). Flu wasused as positive control. IPS as well as peptide pulsed T2 cells, whichwere afterwards not pulsed with tyrosinase, were used as negativecontrols. Tyrosinase-specific recognition was investigated by thetyrosinase-specific T-cell clone IVSB in a ⁵¹Cr-release assay ateffector:target ratio of 2.5:1.

FIG. 15. TCR analysis of the FMNL1-PP2-specific T-cell clone SK22. (A)Analysis of the TCR α-chain repertoire of clone SK22 was performed using34 single V alpha segment-specific primers. The primers Vα14 (band at401 bp) and Vα14.1 (band at 459 bp) amplified identical in-frameCDR3-sequences. Control primers were used to amplify the constant chain(547 bp). Additional fuzzy bands did not result in any readablesequence. (B) TCR β-chain analysis was performed using 37 V betasegment-specific primers. The primers Vβ13 (bands at 278 and 632 bp) andVβ14 (bands at 187 and 541 bp) resulted in identical in-frameCDR3-sequences. The primers for Vβ6.2 and Vβ6.3 (bands at 171 and 439bp) resulted in amplified sequences with out-off-frame generearrangements. Additional fuzzy bands did not result in any readablesequence. Control primers were used to amplify the constant chain (351bp). (C) The FMNL1-specific T-cell clone was stained with aFITC-conjugated anti-human Vβ14-specific antibody and a PE-conjugatedanti-human CD8 antibody (right plot). The isotype control is shown inthe left plot.

FIG. 16. Crossreactivity against HLA-A*3303. IFNγ-release wasinvestigated by ELISA to test the FMNL1-PP2-specific T-cell cloneagainst C1R cells transfected with HLA-A*3303 at effector:target ratioof 1:2. C1R cells transfected with HLA-A*0201 were used as positivecontrol and untransfected C1R cells as well as C1R cells transfectedwith HLA*6601 as negative controls. Error bars indicate the standarddeviation of tested duplicates. Results shown are representative for twoexperiments.

EXAMPLES Antigen Selection

Allorestricted peptide-specific T cells should be directed against tumorassociated antigens with specific characteristics. These antigens shouldhave a most possibly restricted expression to the tumor cell to avoidside effects and should play an important role for vitality and growthof the tumor cell to reduce the risk of target down regulation. Wepreviously identified 14 novel tumor associated antigens using the SEREX(serological identification by recombinant expression cloning) approachin CLL (8). These antigens have been extensively tested for theirpotential use as target antigens and we selected a few of them for ourongoing studies. FMNL1/KW13 (AF432213) is a formin related protein inleukocytes and has been first described using the SEREX approach in CLL(8). We investigated the mRNA expression of FMNL1/KW13 usingquantitative RT-PCR. We observed expression of FMNL1 in healthy tissuemostly in PBMC and to a much lesser extent in bone marrow and thymus.However, we have not seen major expression in any healthy tissue (FIG.1). In contrast, FMNL1/KW13 is highly overexpressed in 60% of CLLsamples tested (FIG. 2 a), some acute leukemia samples (FIG. 2 b) andmalignant cell lines (FIG. 3). Developing specific effectors againstpeptides derived from FMNL1/KW13 might be therefore highly useful forthe development of immunotherapies against malignant lymphomas and alsofor the development of antirtheumatic treatment tools based on thealmost exceptional expression of FMNL1/KW13 in leukocytes.

Peptides derived from FMNL1 were selected using prediction algorithmsfor HLA-binding, proteasomal cleavage sites and TAP transportation(13-16). We are also investigating predicted peptide-antigen candidatesof the B cell receptor α-chain (Table 1).

TABLE 1 Peptides derived from FMNL1/KW13 (or FRL)and B cell receptor α-chain selected forgeneration of allorestricted peptide- specific T cells Name SequenceKW13-PP1 VLLEYLAFA (SEQ ID NO: 1) KW13-PP2 RLPERMTTL (SEQ ID NO: 2)KW13-PP6 CVNEIALSL (SEQ ID NO: 3) KW13-PP7 RLRLTESDKL (SEQ ID NO: 4) FRLRLPERMNTL (SEQ ID NO: 5) BCR1 GLQGTYQDV (SEQ ID NO: 6) BCR2 YLGPGCQAL(SEQ ID NO: 7) BCR3 GTYQDVGSL (SEQ ID NO: 8)Generation of Allorestricted Peptide Specific T Cells

T2 cells which are defective in transporters associated withantigen-processing (TAP) can be efficiently loaded with exogenousHLA-A*0201 binding peptides and were pulsed with selected peptides atdifferent concentrations. Mainly concentrations of 10 μM and 1 μM wereused for pulsing. Peripheral blood mononuclear cells (PBMC) fromHLA-A2-negative donors were isolated by Ficoll density gradient. T cellswere negatively isolated using magnetic bead depletion (Dynal). T cellswere then cultured with peptide-pulsed irradiated T2 cells at ratios of1:10 to 1:100. Cytokines as IL-7 (5 ng/ml), IL-15 (5 ng/ml) and IL-2 (50U/ml) were added. Restimulations were performed after 5-7 days usingagain peptide-pulsed T2 cells. Cultured T cells were stained withHLA-tetrameric complexes and anti-CD8 monoclonal antibodies and weresorted using a flow cytometry sorter. Sorted T cells were cloned bylimiting dilution as well as cultured in oligoclonal T cell lines. Theseclones and unstimulated T cell lines were unspecifically restimulatedwith allogeneic feeder cells (PBMC pools) and OKT3. T cells wereanalyzed for their specificity using ⁵¹Cr release assays, ELISA andtetramer staining.

Using this approach we were able to generate allorestrictedpeptide-specific T cells with specificity against the selected peptideKW13-PP2. These T cells also show specific cytotoxicity againstmalignant cell lines, chronic lymphocytic leukaemia cells, acutelymphoblastic leukaemia cells and also activated lymphocytes (FIG.4-12).

The inventors are currently investigating the T cell receptor (TCR)repertoire of the generated T cell lines and clones. One example is theoligoclonal T cell line PP2-PC1, presenting two α-chains and threeβ-chains which are involved in peptide-specific killing ofallorestricted T cells (Table 2). Subcloning of this T cell line hasbeen performed to identify the specific TCR chain responsible forpeptide-specific killing. Clone SC22 shows a singular α-(Vα14J41) andβ-chain (Vβ14J2.5) which is already represented in the T cell linePP2-PC1, demonstrating the fundamental functional role of these twoCDR3-regions in allorestricted FMNL1-PP2-specificity (Table 3).

TABLE 2 T cell receptor analysis of PP2-PC1 demonstratingthe pattern of an oligoclonal T cell line CDR3 region CDR3 regionAlignment for Alignment for cDNA Amino acid Nomenclature V-gene J-genesequence sequence TCR-α-chains: 1. IMGT TRAV38- TRAJ41*01 GCT TAT GAAA Y E N S 2/DV8*01 AAT TCC GGG G Y A L N TAT GCA CTC F AAC TTG(SEQ ID No: 9) (SEQ ID No: 15) Arden et al. Vα14 J41 GCT TAT GAAA Y E N S AAT TCC GGG G Y A L N TAT GCA CTC F AAC TTC (SEQ ID No: 9)(SEQ ID No: 15) 2. IMGT TRAV21*02 TRAJ21*01 GCT GTG AGG A V R L SCTA AGT ::C # F N K F TTC AAC AAA Y F TTT TAG TTT SEQ ID NO: 10SEQ ID No: 16 Arden et al. Vα23 J21 GCT GTG AGG A V R L S CTA AGT ::C #F N K F TTC AAC AAA Y F TTT TAC TTT SEQ ID NO: 10 SEQ ID No: 16TCR-β-chains: 3. IMGT TRBV7-4*01 TRBJ2-3*01 CAG CAG CTT Q Q L I AATT GCG GGA G G P T D GGG CCT ACA T Q Y P GAT ACG CAG SEQ ID NO: 11TAT TTT SEQ ID No: 17 Arden et al. Vβ6.1 Jβ2.3 CAG CAG GTT Q Q L I AATT GCG GGA G G P T D GGG CCT ACA T Q Y F GAT ACG CAG SEQ ID NO: 11TAT TTT SEQ ID No: 17 4. IMGT TRBV7-4*01 TRBJ2-3*01 GCC AGC AGCA S S L L TTA TTG CGG R E G # T GAG GGC CT: D T Q Y F ACA GAT ACGSEQ ID NO: 12 CAG TAT TTT SEQ ID No: 18 Arden et al. 4β6.1 Jβ2.3GCC AGC AGC A S S L L TTA TTG GCG T E G # T GAG GGC CT: D T Q Y FACA GAT ACG SEQ ID NO: 12 CAG TAT TTT SEQ ID No: 18 5. IMGT TRBV27*01TRBJ2-5*01 GCC AGC AGT A S S F L TTT CTG GGG G E T Q Y GAG ACC CAG FTAG TTC SEQ ID NO: 13 SEQ ID No: 19 Arden et al. vβ14 Jβ2.5 GCC AGC AGTA S S F L TTT CTG GGG G E T Q Y GAG ACC CAG F TAC TTC SEQ ID NO: 13SEQ ID No: 19 6. IMGT TCRBV11- TRBJ2-7*01 GCC AGC AGC A S S L A 2*01TTA GCT TTC F G Q G R GGA CAG S Y E Q Y GGG GGC TCC F TAC GAG CAGSEQ ID NO: 14 TAC TTG SEQ ID No: 20 Arden et al. Vβ21 Jβ2.7 GCC AGC AGCA S S L A TTA GCT TTC F G Q G R GGA CAG S Y E Q Y GGG GGC TCC FTAC GAG CAG TAC TTC SEQ ID NO: 14 SEQ ID No: 20

TABLE 3 T cell clone SC22 shows the followingpattern for α- and β-chain (Vα14J41; Vβ14J2.5): Variable CDR3 regionCDR3 region chain cDNA sequence Amino acid sequence vα14J4lGCT TAT GAA AAT A Y E N S G Y A L N (Arden et al.) TCC GGG TAT GCA FCTC AAC TTC (SEQ ID No: 9) (SEQ ID No: 15) Vβ14J2.5 GCC AGC AGT TTTA S S F L G E T Q Y (Arden et al.) CTG GGG GAG ACC F GAG TAC TTCSEQ ID NO: 13 (SEQ ID NO: 19)

TABLE 4 Prediction scores of selected peptides derived from FMNL1 TAP-Proteasomal HLA-A2 binding transportation* cleavage sites† PeptideSYFPEITHY²⁸ BIMAS²⁹ TAP³⁰ TAP³¹ PAProC II³² FMNL1-PP1 26 1620 1/0 0/1+++(3) VLLEYLAFA SEQ ID NO: 1 FMNL1-PP2 24 201 4/0 2/1  ++(2) RLPERMTTLSEQ ID  NO: 2 FMNL1-PP3 25 15 2/2 1/2 +++(2) ELQEQVALL SEQ ID  NO: 22FMNL1-PP4 27 101 2/2 2/1 +++(2) FINIVVHSV SEQ ID NO: 23 FMNL1-PP5 141, 1 1/1 0/0   −(1) QSLDALLEM SEQ ID  NO: 24 FMNL1-PP6 20 7, 7 2/1 1/0 ++(1) CVNEIALSL SEQ ID  NO: 3 FMNL1-PP7 22 20, 4 2/1 2/1  ++(2)RLRLTESDKL SEQ ID  NO: 4 PMNL1-PP8 31 592 2/0 1/1  ++(4) TLLHYLVKVSEQ ID  NO: 21 *Number of favorable versus (/) unfavorable amino acids†Probability of cleavage at the C-terminus: +++ very strong. ++strong, + weak, − improbable, (number of predicted cute within thepeptide)

LITERATURE

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1. An isolated T cell receptor (TCR) that specifically recognizes anantigenic peptide bound to human leukocyte antigen A2 (HLA-A2), whereinthe antigenic peptide has the amino acid sequence of SEQ ID NO: 2.